Diapause is an important escape mechanism from seasonal stress in many insects. A certain minimum amount of time in diapause is generally needed in order for it to terminate. The mechanisms of time-keeping in diapause are poorly understood, but it can be hypothesized that a well-developed neural system is required. However, because neural tissue is metabolically costly to maintain, there might exist conflicting selective pressures on overall brain development during diapause, on the one hand to save energy and on the other hand to provide reliable information processing during diapause. We performed the first ever investigation of neural development during diapause and non-diapause (direct) development in pupae of the butterfly Pieris napi from a population whose diapause duration is known. The brain grew in size similarly in pupae of both pathways up to 3 days after pupation, when development in the diapause brain was arrested. While development in the brain of direct pupae continued steadily after this point, no further development occurred during diapause until temperatures increased far after diapause termination. Interestingly, sensory structures related to vision were remarkably well developed in pupae from both pathways, in contrast with neuropils related to olfaction, which only developed in direct pupae. The results suggest that a well-developed visual system might be important for normal diapause development.

When females mate with multiple partners, the risk of sperm competition depends on female mating history. To maximize fitness, males should adjust their mating investment according to this risk. In polyandrous butterflies, males transfer a large, nutritious ejaculate at mating. Larger ejaculates delay female remating and confer an advantage in sperm competition. We test whether male ejaculate size in the butterfly Pieris napi (Lepidoptera) varies with female mating history and thus sperm competition, and whether males assess sperm competition using the male-transferred anti-aphrodisiac methyl salicylate (MeS) as a cue. Both sexes responded physiologically to MeS in a dose-dependent manner. Males, however, were more sensitive to MeS than females. Ejaculates transferred by males mating with previously mated females were on average 26% larger than ejaculates transferred by males mating with virgin females, which conforms to sperm competition theory and indicates that males tailored their reproductive investment in response to sperm competition. Furthermore, ejaculates transferred by males mating with virgin females with artificially added MeS were also 26% larger than ejaculates transferred to control virgin females. Male-transferred anti-aphrodisiac pheromone not only functions as a male deterrent, but also carries information on female mating history and thus allows males to assess sperm competition.

Among insects, sexual pheromones are typically mixtures of two to several components, all of which are generally required to elicit a behavioural response. Here we show for the first time that a complete blend of sexual pheromone components is needed to elicit a response also in a butterfly. Males of the Green-veined White, Pieris napi, emit an aphrodisiac pheromone, citral, from wing glands. This pheromone is requisite for females to accept mating with a courting male. Citral is a mixture of the two geometric isomers geranial (E-isomer) and neral (Z-isomer) in an approximate 1:1 ratio. We found that both these compounds are required to elicit acceptance behaviour, which indicates synergistic interaction between processing of the isomers. Using functional Ca2+ imaging we found that geranial and neral evoke significantly different but overlapping glomerular activity patterns in the antennal lobe, which suggests receptors with different affinity for the two isomers. However, these glomeruli were intermingled with glomeruli responding to ,for example, plant-related compounds, i.e. no distinct subpopulation of pheromone-responding glomeruli as in moths and other insects. In addition, these glomeruli showed lower specificity than pheromone-activated glomeruli in moths. We could, however, not detect any mixture interactions among four identified glomeruli, indicating that the synergistic effect may be generated at a higher processing level. Furthermore, correlations between glomerular activity patterns evoked by the single isomers and the blend did not change over time.